The goal of this study is to characterize how neuronal synapses communicate using the Drosophila glutamatergic neuromuscular junction as a model system. Neurotransmitter release following synaptic vesicle (SV) fusion at active zones is the fundamental mechanism for presynaptic communication at synapses. Evoked release is characterized by a synchronous phase of SV fusion that occurs within milliseconds, and a slower asynchronous component that can last for hundreds of milliseconds depending on the neuronal population and firing pattern. In Aim 1, we propose to define how the Synaptotagmin (Syt) Ca2+ sensor family regulates SV fusion. Although many molecular players involved in neurotransmitter release have been identified, how they work mechanistically to control fusion is still unclear. Genetic analysis of Syt 1 has confirmed that it is responsible for sensing Ca2+ influx and driving synchronous fusion of SVs, while also suppressing asynchronous release. In addition to Syt 1, the Syt 7 isoform has been suggested to function as the asynchronous Ca2+ sensor, although this model is still controversial. We will use a structure-function approach to characterize how these two Ca2+ sensors regulate SV fusion. Using a large collection of new point mutants we have generated in Syt 1, we will test how Syt dimerization, docking onto the plasma membrane, and interactions with Complexin and the SNARE complex regulate SV trafficking and fusion. Using Syt 7 and Syt 1/Syt 7 double mutants we will also determine if Syt 7 functions as the asynchronous Ca2+ sensor, or may instead regulate SV availability during high frequency firing. In contrast to SV trafficking, we know little about the regulation of postsynapti vesicle fusion and how retrograde signals modulate synapse biology. In Aim 2, we will characterize the role of the postsynaptic Syt isoform, Syt 4, in regulating Ca2+-regulated vesicle trafficking that underlies retrograde signaling. We have identified Syntaxin 4 as the essential t-SNARE for postsynaptic exocytosis and have shown that the two proteins regulate acute changes in both synaptic structure and function. We will employ APEX labeling techniques and genetic analysis of Syt 4-pHlourin trafficking to define and characterize the postsynaptic vesicle proteome, allowing us to develop a more complete picture of postsynaptic vesicle trafficking and how it contributes to synaptic signaling. The proposed studies will provide important insights into how synapses use vesicular trafficking pathways to mediate bi-directional synaptic communication, providing a foundation to understand how neurological and psychiatric diseases may disrupt these essential elements of neuronal signaling.